Topoisomerases are essential for the orderly synthesis of nucleic acids and thus for cell survival. These enzymes have come to prominence because of their critical role in metabolism and because they are the target for clinically important antibiotic and antitumor drugs. Previous work from this laboratory has shown that potent and specific inhibitors yield valuable insights into the normal function of topoisomerases in trypanosomes; that topoisomerase inhibitors cause profound alterations in DNA structure and replication; and that the severity of these lesions correlates with cytotoxicity. Recent studies reveal that topoisomerase I inhibitors are highly active against Trypanosoma brucei in vitro, with EC50 values in the low nanomolar range. Three projects are proposed. First is to open a new line of investigation into the function of topoisomerase I in trypanosomes. Using a strategy that exploits the drug- promoted covalent linkage of topoisomerase I to kinetoplast DNA networks, a mitochondrial topoisomerase I will be purified and characterized. A gene for T. brucei topoisomerase I will be isolated by means of phylogenetically conserved sequences. The effects of topoisomerase I inhibitors on the synthesis and structure of kDNA will be studied, and topoisomerase I cleavage site will be mapped. The second project will be a continuation of ongoing topoisomerase II studies. Specifically, a nuclear topoisomerase II will be isolated and immunolocalized, possibly to distinct spatial regions of the nucleus. Drug-promoted topoisomerase II cleavage sites in nuclear DNA will be mapped, to determine their location and abundance in transcribed vs inactive (minichromosome) regions. The third project is designed to identify structural features of topoisomerase inhibitors that enhance cytotoxic activity against trypanosomes in vitro, and to ascertain the relative contributions of nuclear and mitochondrial topoisomerases to cytotoxicity. The inhibitors that appear most promising in vitro will be tested in mice. This work will increase our understanding of the often unique nucleic acid metabolism of trypanosomes; it may also provide a clear avenue to much- needed new therapy for trypanosomiasis.